key: cord-0699749-a6unp81j authors: Tada, Takuya; Zhou, Hao; Dcosta, Belinda M.; Samanovic, Marie I.; Chivukula, Vidya; Herati, Ramin S.; Hubbard, Stevan R.; Mulligan, Mark J.; Landau, Nathaniel R. title: Increased resistance of SARS-CoV-2 Omicron Variant to Neutralization by Vaccine-Elicited and Therapeutic Antibodies date: 2021-12-30 journal: bioRxiv DOI: 10.1101/2021.12.28.474369 sha: 9df5dfd8467c337aef27b885be441211b296bd61 doc_id: 699749 cord_uid: a6unp81j Currently authorized vaccines for SARS-CoV-2 have been highly successful in preventing infection and lessening disease severity. The vaccines maintain effectiveness against SARS-CoV-2 Variants of Concern but the heavily mutated, highly transmissible Omicron variant poses an obstacle both to vaccine protection and monoclonal antibody therapies. Analysis of the neutralization of Omicron spike protein-pseudotyped lentiviruses showed a 26-fold relative resistance (compared to D614G) to neutralization by convalescent sera and 26-34-fold resistance to Pfizer BNT162b2 and Moderna vaccine-elicited antibodies following two immunizations. A booster immunization increased neutralizing titers against Omicron by 6-8-fold. Previous SARS-CoV-2 infection followed by vaccination resulted in the highest neutralizing titers against Omicron. Regeneron REGN10933 and REGN10987, and Lilly LY-CoV555 and LY-CoV016 monoclonal antibodies were ineffective against Omicron, while Sotrovimab was partially effective. The results highlight the benefit of a booster immunization in providing protection against Omicron but demonstrate the challenge to monoclonal antibody therapies. The vaccines that have been granted emergency use authorization (EUA) have proven highly 42 protective against SARS-CoV-2, resulting in a major decrease in infection rates, hospitalization 43 and deaths [1] ; however, the appearance of recently evolved viral variants classified as variants 44 of concern (VOC) [2] that contain multiple mutations in the viral spike protein have raised 45 concerns about potential decreases in vaccine effectiveness. These concerns have been 46 assuaged by laboratory findings of modest 2-5-fold decreases in neutralizing antibody titer 47 against the VOCs [2-7] and epidemiological evidence of continued vaccine protection [8, 9] . Antibody neutralization assay 140 Sera or monoclonal antibody was serially two-fold diluted and then incubated with an amount of 141 virus corresponding to a volume that resulted in MOI=0.2 on ACE2.293T or Vero cells for 142 pseudotyped virus. After 30-minute incubation at room temperature, the virus was added to 1 X 143 10 4 target cells in a 96 well culture dish. The cells were cultured for 2 days after which the 144 culture medium was removed and 50μl Nano-Glo luciferase substrate (Nanolight) was added. Luminescence was read in an Envision 2103 microplate luminometer. All samples were tested in duplicate or triplicate. Data were analyzed using GraphPad Prism 8 149 software and statistical significance was determined by the two-tailed unpaired t-test or 150 nonparametric ANOVA test. Significance was based on two-sided testing and attributed to p< To determine the effectiveness of antibodies induced by infection with earlier SARS-CoV-2 159 variants to protect from re-infection with the Omicron variant, we tested neutralizing antibody 160 titers in the sera of unvaccinated participants involved in an ongoing clinical study that had been 161 collected 32 to 57 days post-COVID-19 symptom onset. Neutralizing antibody titers were 162 measured using lentiviral virions pseudotyped by the parental D614G, Alpha, Beta and Delta 163 spike proteins, an assay that accurately reflects titers obtained in the plaque reduction 164 neutralization test (PRNT). The results showed modest reductions in neutralizing titer against 165 Beta and Delta as compared to the parental D614G but a more substantial average 26-fold 166 reduction in titer against Omicron. Approximately 60% of the donor sera had titers below the IC50 of 20 limit of detection in the assay (Fig. 1A) . To determine the effectiveness of antibodies 168 elicited by vaccination, we tested sera collected 70 days post-immunization from study 169 participants who had been fully vaccinated (two immunizations) with BNT162b2 or Moderna 170 mRNA-1273 mRNA vaccines (Fig. 1B) . Titers against the D614G virus were 3-4-fold higher 171 than those of the convalescent patient sera and the general pattern of neutralization of the 172 variants was similar. Notably, neutralizing antibody titers against the Omicron pseudotype was 173 decreased 26-34-fold compared to D614G. Previous infection has been shown to strengthen and broaden the neutralizing antibody 176 response to SARS-CoV-2 variants upon vaccination. To determine whether previous infection would increase neutralizing antibody titers against the Omicron variant, we tested sera from study participants who were vaccinated with BNT162b2 and had, or had not, been previously 179 infected with SARS-CoV-2 (Fig. 1C) . Sera from study participants without previous infection, 180 collected one month post-second vaccination, had high titers of neutralizing antibody against 9 1.4-fold and against Omicron 18-fold. Titers were had only slightly declined 7-8 months post-183 vaccination. One-month post-boost, titers increased for all variants. Titers against Omicron 184 remained 14-fold lower than against D614G. Notably, study participants who had poor 185 neutralizing titers against Omicron after two immunizations had increased their titers following 186 the boost (Fig. 1D) . Sera from previously infected study participants were on average 3-4-fold 187 higher than those without previous infection and had a similar ratio of neutralizing titers among 188 the different variants. Sera from previously infected study participants post-boost achieved high 189 neutralizing titers against the Beta and Delta variants. While titers against Omicron also rose, Mapping of the amino acid residues responsible for the escape from the monoclonal antibodies 293 showed that most of the mutations had no effect but that several had partial effects on Etesevimab. The findings suggest that the Regeneron and Eli Lilly cocktails will not be effective 303 for the treatment of patients infected by the Omicron variant. The effectiveness of Sotrovimab 304 cannot be predicted from these data but it would seem likely that it will not be as effective on 305 patients infected with Omicron variant as compared to those with Delta or other variants. Our findings suggest that while the frequency of infections with the Omicron variant are likely to 308 increase, the titers achieved by full vaccination followed by a booster immunization will protect 1 4 most individuals from developing severe disease. The T cell response induced by vaccination, 310 which is less prone to immune escape, may also provide additional protection. Our findings 311 provide further support for the benefits of booster immunization and point to the need to develop 312 additional therapeutics for the treatment of COVID-19. The emergence of the Omicron variant raises concern about the possibility of additional 315 evolutionary leaps for the virus and the need to preempt any such variants before they emerge. While the current surge in Omicron infections may increase hospitalization and mortality, there 317 is also an increased likelihood of leading to herd immunity that may protect against future New data show treatment with Lilly's neutralizing antibodies bamlanivimab CoV555) and etesevimab (LY-CoV016) together reduced risk of COVID-19 451 hospitalizations and death by 70 percent Efficacy and safety of two neutralising monoclonal antibody 453 therapies, sotrovimab and BRII-196 plus BRII-198, for adults hospitalised with COVID-19 454 (TICO): a randomised controlled trial CoV-2 variant: Unique features and their impact on pre-existing antibodies An ACE2 Microbody Containing a Single Immunoglobulin 460 Fc Domain Is a Potent Inhibitor of SARS-CoV-2 Neutralizing antibody levels are highly 462 predictive of immune protection from symptomatic SARS-CoV-2 infection Immune correlates analysis of the 465 mRNA-1273 COVID-19 vaccine efficacy clinical trial Outpatients with Covid-19 REGN-COV2 antibodies prevent and treat SARS